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1
1.
. () 3-5
DNA, .
.
DNA 4-8 .
.
DNA (.. DNA ).
. DNA ()
.
EcoRI
E. coli,
5 G-A--T-T-C 3
3 C-T-T-A-A-G.5
DNA G
5-A-A-T-C-G-C-G-T-G-G-A--T-T-C-A-A-T-C-G-C-G-G-A-G-3.
3-T-T-A-G-C-G-C-A-C-C- T-T-A-A-G-T-T-A-G-C-G-C-C-T-C-5
,
( ).
5-A-A-T-C-G-C-G-T-G-G -A--T-T-C-A-A-T-C-G-C-G-G-A-G-3
+
3-T-T-A-G-C-G-C-A-C-C- T-T-A-A G-T-T-A-G-C-G-C-C-T-C-5
. .
() 5 3 DNA.
,
2
2.
DNA () .
* DNA,
.
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3.
DNA
DNA .
(. DNA )
:
.
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DNA DNA
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) .
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4.
DNA
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DNA
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. 1
DNA.
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..
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5-A-A-T-C-G-C-G-T-G-G -A--T-T-C-A-A-T-C-G-C-G-G-A-G-3
+
3-T-T-A-G-C-G-C-A-C-C- T-T-A-A G-T-T-A-G-C-G-C-C-T-C-5
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4
(..)
DNA
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DNA. DNA DNA
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DNA
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DNA .
. DNA
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1.
(DNA
RNA), DNA-, .
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DNA 100 C,
DNA .
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DNA:DNA RNA:RNA DNA:RNA
DNA
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-
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9
_
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10
cDNA
,
,
.
,
mRNA
mRNA.
,
mRNA
mRNA ,
.
mRNA ( DNA)
, mRNA DNA
RNA DNA.
(complementary DNA, cDNA).
,
11
cDNA .
. mRNA
. DNA RNA.
. DNA-RNA.
. RNA
. RNA ( ) RNA
( ), DNA
cDNA
. DNA
.
cDNA mRNA .
------------------------------------------------------------------------------------------------------------------------
cDNA :
m RNA.
tRNA, rRNA, snRNA
---------------------------------------------------------------------------------------------------------------------------
,
12
_ cDNA
__
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--
1. ; 2. ; 3. ; 4.
, ;
5. ; 6. cDNA . 7.
cDNA ; 8. . 9. in vitro. 10.
; 11. DNA. 12. ; 13.
5 AAGCTTCCATGAAGTTCAAAGAATTCTTTCCC3 EcoRi
14. DNA
5 AATCTCGAATTCAA.TTACTTAAGGTT 3 3 TTAGAGCTTAAGTT.AATGAATTCCAA..5
EcoRI; 15.
210 . , ;
16.
.
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14
1
.. 1. :
. . . . .
2. 3-5 :
. . . DNA .
3. ;
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4. :
. . . DNA . DNA RNA
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6. :
. mRNA . DNA . . . DNA
7.
DNA: . DNA . . DNA .
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15
8. :
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9. EcoRI:
. . GAATTC DNA . . GAATTC DNA
10. :
. DNA . DNA RNA . DNA DNA RNA . .
() ()
1. DNA. 2. . 3. PCR in vitro . 4. DNA . 5.
. 6. 5GAATTC- 3 . 7. . 8.
. 9. ,
. 10. PCR DNA
.
11 22
. 1.
.
2.
. 3.
. PCR 4.
. cDNA 5. in vitro
6. 3-5
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16
1.
DNA:
SmaI CCC GGG
GGG CCC
Notl GC GGCCGC
CG CCGG CG
2.
, .
E. coli .
EcoRI
.
DNA
EcoRI.
E. coli
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.
3. DNA ( EcoR),
: ()
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EcoR.
DNA
:
(1) (2) .
DNA;
,
17
4. : () DNA () () , . EcoRI. , BamHI, NotI, HindIII . 1) ; 2)
DNA;
()
()
5. DNA ,
, 1000 , ,
300 .
)
;
) DNA
DNA .
;
,
18
6. BamHI .
G GATCC CCTAG G DNA BamHI . GATC Sau3A N GATCN N CTAG N
) BamHI Sau3A DNA )
7. AACGAATTCGATGGATCCA..602.....GGATCCGTAGAATTCACC EcoRI GAATTC HambIII GGATCC CTTAAG CCTAGG : () ; () mRNA; () ; () ;
8. PCR 3000 30
) 30 ; ) ; ( 5 min).
,
19
.
1. , -----------------------
2. ,
-----------------------
3. DNA ----------------------
4. DNA
---------------------
5. DNA ----------------------
6. DNA, ------------ ------------ ------------, DNA
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7. ----------------
---------------------- DNA
8. --------------------- ---------
--------
9. -----------
10. DNA
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. -
1.
DNA
2. ,
3. DNA
4.
DNA
5. DNA
,
20
6. DNA DNA
.
7.
DNA
8. EcoRI GAATTC G
9. RNA
10. PCR DNA in
vitro.
.
cDNA
mRNA-cDNA. cDNA
:
..TTATGGGTACTTTGAGCCCAAAACTATAGTATT
cDNA ,
)
) .
,
21
: DNA, : :
:
) DNA :
_____________________________
) , : __________________
) DNA DNA ,
DNA :
_________________________
) DNA ,
: ___________________________
)
mRNA :
___________________________
) RNA DNA :
_____________________
) DNA
, :
___________________________
:
) in vivo
in vitro
) -
;
) cDNA
;
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22
)
DNA;
DNA;
) cDNA ;
.
) DNA (.) EcoRI
2 . DNA .. HindIII
3 .
.. ; DNA
;
) DNA 10000
:
DNA % % %C %G
10 20 20 30 30
20 5 5 45 45
;